Host–Symbiont Specificity Determined by Microbe– Microbe Competition in an Insect Gut

Host–symbiont specificity determined by microbe– microbe competition in an insect gut

Hideomi Itoha,1, Seonghan Jangb,1, Kazutaka Takeshitac, Tsubasa Ohbayashid, Naomi Ohnishie,2, Xian-Ying Mengf, Yasuo Mitania, and Yoshitomo Kikuchia,b,g,3

aBioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Hokkaido Center, 062-8517 Sapporo, Japan; bGraduate School of Agriculture, Hokkaido University, 060-8589 Sapporo, Japan; cFaculty of Bioresource Sciences, Akita Prefectural University, 010-0195 Akita, Japan; dInstitute for Integrative Biology of the Cell, UMR 9198, CNRS, Commissariat à l’Energie Atomique et aux Énergies Alternatives (CEA), Université Paris-Sud, 91198 Gif-sur-Yvette, France; eResearch Center for Zoonosis Control, Hokkaido University, 001-0020 Sapporo, Japan; fBioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba Center, 305-8566 Tsukuba, Japan; and gComputational Bio Big Data Open Innovation Laboratory, National Institute of Advanced Industrial Science and Technology, 062-8517 Sapporo, Japan

Edited by Joan E. Strassmann, Washington University in St. Louis, St. Louis, MO, and approved September 30, 2019 (received for review July 18, 2019) Despite the omnipresence of specific host–symbiont associations microbe competition on the evolution and stabilization of host– with acquisition of the microbial symbiont from the environment, symbiont specificity is very scarce. little is known about how the specificity of the interaction evolved The bean bug Riptortus pedestris (Heteroptera: Alydidae) is and is maintained. The bean bug Riptortus pedestris acquires a associated with a Burkholderia symbiont that is confined in specific bacterial symbiont of the genus Burkholderia from environ- symbiosis-specific crypts located in the posterior midgut region Burkholderia mental soil and harbors it in midgut crypts. The genus M4 (10). The Burkholderia symbiont is not essential but benefi- consists of over 100 species, showing ecologically diverse lifestyles, cial for growth, reproduction, immunity homeostasis, and pesti- and including serious human pathogens, plant pathogens, and cide resistance of the insect host (11–13). While most insects nodule-forming plant mutualists, as well as insect mutualists. vertically transmit their symbionts, bean bugs acquire their mi- Through infection tests of 34 Burkholderia species and 18 taxonomically diverse bacterial species, we demonstrate here that non- crobial partners from the ambient soil in every generation (10, 11). To acquire their specific symbionts from the diverse soil symbiotic Burkholderia and even its outgroup Pandoraea could EVOLUTION stably colonize the gut symbiotic organ and provide beneficial microbiota, stinkbugs appear to utilize a specific organ called the effects to the bean bug when inoculated on aposymbiotic hosts. “constricted region,” a narrow passage filled with a mucus-like However, coinoculation revealed that the native symbiont always matrix, located in the midgut immediately upstream of the crypt- outcompeted the nonnative bacteria inside the gut symbiotic or- bearing region. The constricted region winnows out contami- gan, explaining the predominance of the native Burkholderia sym- nating microorganisms and the Burkholderia symbiont specif- biont in natural bean bug populations. Hence, the abilities for ically penetrates into the symbiotic region (14). In addition to colonization and cooperation, usually thought of as specific traits this specific apparatus for bacterial sorting, the insect expresses of mutualists, are not unique to the native Burkholderia symbiont diverse antimicrobial peptides in the symbiotic organ (13, 15), but, to the contrary, competitiveness inside the gut is a derived which may also play a role in the partner choice. trait of the native symbiont lineage only and was thus critical in the evolution of the insect gut symbiont. Significance

gut symbiosis | symbiont specificity | stinkbug | Burkholderia | competitiveness How are specific host-symbiont mutualisms stabilized without vertical transmission? This is one of the fundamental questions in evolutionary biology. To ensure specificity, animals and plants number of animals and plants are associated with beneficial have evolved sophisticated sorting mechanisms. Theoretical Amicroorganisms, which provide diverse services to the host studies proposed another mechanism, so-called “competition- species, such as enhanced nutrition and protection from antag- based selection,” where hosts provide a specific environment onists (1, 2). While some symbionts are vertically transmitted to selectively cultivate favorable symbionts and maintain sym- from mother to offspring, many animals and plants acquire in biont quality. Although this mechanism is thought to be powerful every generation the symbionts from the environment (3). Since in open systems, such as gut symbiosis where contaminants microorganisms are abundant and diverse in the environment, regularly invade, until now little experimental evidence has sup- hosts have evolved elaborate mechanisms for “partner choice” to ported its importance in the evolution of symbioses. Through the ensure the specificity of the associations (4, 5). Well-studied inoculation of an insect host with a large panel of symbiotic and model systems, the legume–Rhizobium and squid–Vibrio symbi- nonsymbiotic bacteria, we demonstrate that microbial competi- oses, have revealed that the partner specificity is achieved by tion is critical to maintain the specificity in an insect gut symbiosis. various host mechanisms, such as signal recognition during initiation and secretion of antimicrobial agents during colonization Author contributions: H.I., S.J., and Y.K. designed research; H.I., S.J., T.O., N.O., X.-Y.M., Y.M., and Y.K. performed research; H.I., S.J., K.T., and Y.K. analyzed data; and H.I., S.J., (6, 7). In addition to such host control mechanisms, theoretical and Y.K. wrote the paper. – modeling studies have suggested the importance of microbe The authors declare no competing interest. microbe competition to ensure the specificity, wherein the host This article is a PNAS Direct Submission. provides a demanding environment to let a favorable microor- Published under the PNAS license. “ ganism grow dominantly (8, 9). The competition-based selec- 1H.I. and S.J. contributed equally to this work. tion” is thought to be more powerful to eliminate contaminants 2Present address: Cancer Precision Medicine Center, Japanese Foundation for Cancer Re- or pathogens and maintain symbiont quality, particularly in open search, 135-850 Koto-ku, Japan. systems, such as the gut, the fungus-farming garden of insects, and 3To whom correspondence may be addressed. Email: [email protected]. the plant rhizosphere, where microbial contamination can regu- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. larly occur and potentially disrupt the symbioses. Despite these 1073/pnas.1912397116/-/DCSupplemental. theoretical studies, empirical support for the impact of microbe–

www.pnas.org/cgi/doi/10.1073/pnas.1912397116 PNAS Latest Articles | 1of10 Downloaded by guest on September 30, 2021 To be a bean bug symbiont, therefore, the Burkholderia species Burkholderia cepacia, Burkholderia pseudomallei and Burkholderia should be capable of penetrating the constricted region (initiation), mallei, designated as the “B. cepacia complex and B. pseudomallei” stably colonizing the symbiotic organ (accommodation), and of (BCC&P) clade (22). Some BCC&P species are also reported as course, behaving cooperatively with the host (cooperation). Re- fungal symbionts (23) and beetle symbionts (24). The second clade cent studies have revealed part of the symbiont’s mechanisms in- includes a number of plant growth-promoting rhizobacteria and volved in these processes. The Burkholderia symbiont employs a nodule symbionts of leguminous plants, and is designated as the unique corkscrew flagellar motility to pass through the constricted “plant-associated beneficial and environmental” (PBE) clade (25). region (16); it undergoes cytological and metabolic alterations, This clade, which also includes farming symbionts of the slime mold such as flagella loss and polyhydroxyalkanoate accumulation, to Dictyostelium discoideum (26), was recently even nominated as a cope with the crypt environment (17–19); and it rapidly prolifer- new genus, Paraburkholderia (27). The third clade mainly consists ates to occupy entirely the available space in the lumen of the of environmental species, leaf-nodule symbionts of Rubiaceae plants, symbiotic M4 midgut region, notably by recycling host metabolic and gut symbionts of stinkbugs, and is called the “Burkholderia wastes into essential amino acids and B vitamins in the M4 crypts, ” “ which are thought to be part of the symbiont services provided to glathei clade or the stinkbug-associated beneficial and envi- ” the host (17). ronmental (SBE) clade (10, 28). Also for this clade, a new The bacterial genus Burkholderia (Betaproteobacteria: genus name, Caballeronia, was proposed (29). The outgroup of Burkholderiaceae), comprises over 100 species with taxonomically Burkholderia is the genus Pandoraea (Fig. 1), which are common validated names and is an ecologically very diverse bacterial group soil bacteria, although some of them are opportunistic pathogens (20). Based on genomic phylogeny, the genus Burkholderia is of humans (30). Almost all of the Burkholderia and Pandoraea grouped into at least 3 distinct clades (21) (Fig. 1). The first clade species, except the leaf-nodule symbionts in SBE, are easy to consists of many human-, animal-, and plant-pathogens, including culture and genetically manipulable, providing us with an

Fig. 1. Phylogenetic relationship of 113 species of the genus Burkholderia. A multilocus tree based on 6,399 unambiguously aligned amino acid sites of 21 genes is shown. The 21 marker genes were commonly present in the 113 Burkholderia species and 4 betaproteobacteria with well-conserved alignment brocks (>60% of the alignment). Stinkbug-associated SBE, plant-associated PBE, and pathogenic BCC&P groups are highlighted with blue, green, and red, respectively. Recently proposed new genus names of SBE and PBE are also depicted under the group names. Closed circles indicate that the nodes are supported with >70% bootstrap values. Illustrations courtesy of Chiaki Matsuura and Yu Matsuura.

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1912397116 Itoh et al. Downloaded by guest on September 30, 2021 opportunity to clarify how symbiotic traits have evolved in a phylogenetically well-defined bacterial lineage. Genetically diverse Burkholderia species or strains are associated with natural R. pedestris populations but nearly all of them belong to the SBE clade (11, 31, 32) (SI Appendix, Fig. S1) and only 1 study reported the infection with BCC&P species in overwintering R. pedestris (33). In addition, in insects reared on soil in the laboratory, we occasionally identified infections with PBE Burkholderia as well as Pandoraea, suggesting that colonization of the symbiotic midgut organ is not strictly limited to SBE species (34). Although we revealed that the bacterial sorting organ in the bean bug gut winnows out symbionts from nonsymbionts, only a limited number of bacterial species (i.e., Eschelichia coli, Pseudomonas putida,andBacillus subtilis)have been tested as nonsymbionts (14). Here we show by experimental inoculations of diverse bacterial species into R. pedestris that non-SBE Burkholderia and even Pandoraea are capable of passing through the constricted region, stably colonizing the gut symbiotic organ, and behaving cooperatively. We further demonstrate that, through competitive infection assays in conjunction with histological inspections, SBE always outcompetes these nonnative symbionts inside the gut symbiotic organ, highlighting a pivotal role of symbiont competitiveness for stabilizing the insect– microbe gut symbiosis. Results and Discussion SBE, PBE, and Pandoraea Species Can Pass through the Constricted EVOLUTION Region and Stably Colonize the Symbiotic Organ. The midgut of R. pedestris consists of 4 distinct sections called M1, M2, M3, and M4. The SBE Burkholderia specifically colonizes the crypt-bearing M4 section. After symbiont infection, the M4 crypts swell and become whitish in color because of the symbiont colonization and proliferation in their lumen (10). In front of M4, a bulbous area called the M4 bulb (M4B) develops, wherein symbiont cells, flowed backward from M4, are entirely digested and the derived nutrients absorbed by the host (15, 17, 35). Probably because of the digested bacteria, the M4B becomes swollen and yellowish (10). The constricted region connects the M3 with the M4B (14). In order to analyze further the infection specificity of the stinkbug–Burkholderia gut symbiosis, we first experimentally inoculated 20 taxonomically diverse bacterial species of 3 major phyla into the bean bug and determined their colonization ability by counting colony forming units (CFU). The tested bacteria (SI Appendix, Table S1) included species of the Burkholderia (SBE [Burkholderia insecticola], PBE [Burkholderia fungorum], and BCC&P [Burkholderia plantarii]), related species of the family Burkholderiaceae (Ralstonia, Chitinimonas, Cupriavidus,andPandoraea species), as well as more distantly related species of the Alpha- and Gammaproteobacteria. Inoculation was performed in early second instar when the insects are competent for symbiont acquisition (36), and bacterial colonization was investigated at day 2 of the third instar (i.e., 5 d postinoculation), which is 2 to 3 d after completion of the colonization process with B. insecticola (37). Whereas most of the tested species showed little or no infection in Fig. 2. Infection specificity of bacterial species in the gut symbiotic organ. the symbiotic organ, the PBE and Pandoraea, as well as the SBE (A) Infection ability of diverse bacterial species to the gut symbiotic organ. but not the BCC&P, colonized the symbiotic organ to high density, Five days after oral administration, the colonization level in M4 was evalu- around 107 CFU per organ (Fig. 2A). Microscopic observations ated by CFU counting. (B) Infection initiation process visualized by confocal microscopy. The infecting bacteria were labeled with GFP. Note that only with green fluorescent protein (GFP)-labeled strains of E. coli, Pandoraea (P. norimbergensis), PBE (B. fungorum), and SBE (B. insecticola) Burkholderia, and allied Burkholderiaceae species confirmed that pass through the sorting organ, the constricted region. Arrows indicate while E. coli, Cupriavidus, and BCC&P reached the M3 but did an infection thread invading from M3 to M4B through the constricted re- not pass through the constricted region, the SBE, PBE, and gion. Abbreviations: CR, constricted region; M3, midgut third section; Pandoraea species could pass through the constricted region and M4B, M4 bulb. penetrate into the symbiotic M4 region (Fig. 2B).

Infection States of Burkholderia and Pandoraea Species in the Bean morphology of infected midguts were surveyed for 13 SBE species, Bug. To further detail the specificity of the interaction and to 12 PBE species, 7 BCC&P species, 2 species of other Burkholderia clarify the infection states of Burkholderia and Pandoraea species clades, and 2 Pandoraea species. Results of the infection assays, in R. pedestris, infection rate (percent of infected insects) and morphometric measurements, and microscopic observations, are

Itoh et al. PNAS Latest Articles | 3of10 Downloaded by guest on September 30, 2021 summarized in Fig. 3 and SI Appendix, Fig. S2 and Table S2,re- M4 crypts were infected but not well bulged (SI Appendix,Fig. spectively. Details of the infection tests are described in the fol- S2 B and C); the M4B was significantly larger (SI Appendix,Fig. lowing sections and shown in SI Appendix, Figs. S3–S10. S2D) but remained whitish and did not adapt the yellowish SBE Burkholderia. All of the 13 tested SBE Burkholderia species or color characteristic for SBE infection (SI Appendix,Fig.S2E). strains consistently showed high, 80 to 100%, infection rates (SI LSM and TEM observations revealed that this nonnative sym- Appendix, Table S2). In aposymbiotic insects, M4 crypts and biont occupied strongly the central tract of the M4 but had only M4B were not well developed (Fig. 3A), the luminal space of partially colonized the crypt lumen (Fig. 3C and SI Appendix, each crypt was small, and its epithelium cells were thick (Fig. Fig. S2F). When colonized, the luminal space of each crypt was 3A). In contrast, the symbiotic organ infected with SBE (B. narrower and its epithelial cells were thicker than those of the insecticola) was well developed (Fig. 3B and SI Appendix,Fig. crypts colonized by SBE (Fig. 3 B and C). Colonizing PBE cells S2): M4 crypts were bulging by the bacterial proliferation and had a typical rod shape and were not notably different from colonization (SI Appendix, Fig. S2 B and C); the M4B became colonizing SBE cells (SI Appendix,Fig.S3). Although no or bulbous in shape (SI Appendix, Fig. S2D) and yellowish in color partial colonization of SBE Burkholderia occurredintheM4B, (SI Appendix, Fig. S2E). Laser-scanning microscopy (LSM) and TEM observations showed that a high number of bacterial cells transmission electron microscopy (TEM) (21) observations con- of B. fungorum colonized this region (SI Appendix,Fig.S4), firmed that M4 crypts were entirely filled with SBE cells (Fig. 3B suggesting that the PBE is digestion-tolerant or that the digesting and SI Appendix, Fig. S3), while no or partial colonization oc- enzymes are not properly produced and that the colonizing cells curred in the M4B (SI Appendix, Fig. S4). These morphological cause the swelling and whitish color of M4B. These morphological features of M4 and M4B were consistently observed in insects features (i.e., smaller M4 crypts and swollen and whitish M4B) infected with the other 12 species of SBE Burkholderia (SI Ap- were consistently observed in insects infected with other PBE pendix, Figs. S5 and S9 and Table S2). species (SI Appendix, Fig. S6 and Table S2). PBE species with low PBE Burkholderia. Among the 12 tested species of PBE Burkholderia, infection rates did not alter morphogenesis of M4B and M4, which the infection rate was variable (SI Appendix,TableS2). Five resembled the aposymbiotic state. Thus, the infection rate and species showed a very high (90 to 100%) infection rate, while morphological alterations in M4 and M4B were highly correlated 4 species showed moderate (40 to 70%) and 3 species showed (SI Appendix, Fig. S9 D and E), suggesting that bacterial in- low (0 to 10%) infection rates. The morphology of the symbiotic fection and proliferation in the gut symbiotic organ stimulate the organ infected with the highly infective representative, B. fungorum, morphological change. was observed in detail. This revealed that the symbiotic organ BCC&P Burkholderia and other Burkholderia species. The 7 tested colonized by PBE Burkholderia (Fig. 3C) was markedly differ- BCC&P species, including 2 species of biosafety level 3 (BSL3) ent from the organ colonized by SBE Burkholderia:The pathogens, B. mallei and B. pseudomallei, consistently showed a

Fig. 3. Morphological alteration of the gut symbiotic organ after colonization of symbiotic and nonsymbiotic bacteria. Images of whole midgut, M4B and M4 region, LSM of M4, and TEM of M4 in an aposymbiotic insect (A), and insects inoculated with SBE (B), PBE (C), and Pandoraea (D). All images are of third- instar nymphs. Abbreviations: H, hindgut; M1, midgut first section; M2, midgut second section; M3, midgut third section; M4B, M4 bulb; M4, crypt-bearing midgut fourth section. In LSM images: green, SYTOX GREEN staining of colonizing bacteria and host nuclei; red, phalloidin staining of cytoskeleton; yellow arrowheads, infected M4 crypts; white arrowheads, uninfected M4 crypts. In TEM images: lm, luminal region of crypt; n, host nucleus.

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1912397116 Itoh et al. Downloaded by guest on September 30, 2021 low (0 to 40%) infection rate (SI Appendix, Table S2). In this PBE Burkholderia and Pandoraea Are Beneficial for the Bean Bug. The BCC&P group, even when infection was detected by diagnostic inoculation tests revealed that many PBE Burkholderia species PCR, the M4 and M4B were small and not well-developed, as is and even the outgroup, Pandoraea, passed through the con- the case in aposymbiotic insects (SI Appendix, Figs. S7 and S9). stricted region and stably accommodated in the M4 region, al- GFP-labeled Burkholderia glumae and Burkholderia pyrrocinia con- though they did not fully colonize the crypt lumen. To determine firmed that the BCC&P species can penetrate the constricted re- if the PBE and Pandoraea species are beneficial for the bean bug, fitness parameters of the insect host (survival rate, growth period, gion and reach the M4 region (SI Appendix,Fig.S10), although they body size, and weight) were measured after infection. B. insecticola did not stably accommodate inside the crypts. Other Burkholderia (SBE), B. fungorum (PBE), and P. norimbergensis (Pandoraea), all species of independent clades, Burkholderia andoropogonis and of which show a 100% infection rate, were tested. Compared with Burkholderia caryophylli, showed no infection (SI Appendix, Figs. aposymbiotic insects, insects infected with either of these strains S7 and S9 and Table S2). showed significantly higher survival rates, shorter developmental Pandoraea and other Burkholderiaceae. Both tested Pandoraea species, time before adulthood, larger body size, and heavier body weight Pandoraea norimbergensis and Pandoraea oxalativorans,showed (Fig. 4). However, insects infected with the Pandoraea neverthe- a 100% infection rate (SI Appendix,TableS2). Similar to PBE less showed a slower developmental time and smaller body weight Burkholderia, M4 crypts were infected but not properly developed than those infected with the SBE Burkholderia (Fig. 4), while in- and the M4B was strongly swollen in shape but whitish in color sects infected with the PBE species showed an almost identical (Fig. 3D and SI Appendix, Figs. S2 and S8). LSM and TEM ob- body size and weight to those with SBE Burkholderia, although the servations revealed that Pandoraea were abundant in the central growth period was significantly delayed (Fig. 4). Taken together, duct of M4 but only partially colonized the crypt lumen (Fig. 3D the rearing experiments clearly demonstrated that the nonnative and SI Appendix,Fig.S2F). TEM observations revealed that bacteria B. fungorum and P. norimbergensis are not parasitic but M4 colonized cells were typical rods (SI Appendix,Fig.S3)and enhance survivability, growth, and development of the host in- Pandoraea cells densely colonized M4B (SI Appendix,Fig.S4), sect, although to a slightly lesser extend than the native symbiont B. insecticola. similar to PBE Burkholderia, suggesting that the Pandoraea is also digestion-tolerant and the colonizing cells cause the swelling and SBE Burkholderia Always Outcompete PBE and Pandoraea in the Gut

whitish color of M4B. Other Burkholderiaceae species, Cupriavi- Symbiotic Organ in Coinfection Assays. PBE Burkholderia and EVOLUTION dus taiwanensis, Ralstonia solanacearum,andChitimonas koreensis, Pandoraea species are common bacterial groups in soil envi- did not infect the symbiotic organ (SI Appendix, Figs. S8 and S9 ronments, and detected frequently with SBE Burkholderia (34, and Table S2). 38, 39). Given the high infectivity and benefits of PBE and

Fig. 4. Fitness effect of symbiotic and nonsymbiotic bacteria in the bean bug. Comparison of fitness parameters between aposymbiotic insects (Apo) and insects infected with Pandoraea (Pan: P. norimbergensis), PBE (B. fungorum), and SBE (B. insecticola). (A) Survival rate (= adult emergence rate), (B) time to adulthood, (C) photographs of male and female adults. (D–F) Male insects: (D) maximum thorax width, (E) body length, (F) dry weight. (G–I) Female insects: (G) maximum thorax width, (H) body length, and (I) dry weight. Different letters indicate statistically significant differences (P < 0.05). The fitness parameters were analyzed by a Mann–Whitney U test (B and D), Student t test (E and F), Brunner–Munzel test (G), and Welch t test (H and I), and corrected by the Bonferroni method. Error bars indicate SDs. Number of replicates are depicted on the bars.

Itoh et al. PNAS Latest Articles | 5of10 Downloaded by guest on September 30, 2021 Pandoraea, why do only SBE Burkholderia prevail in field pop- that the SBE Burkholderia significantly outcompeted the PBE ulations of the bean bug? To clarify this point, colonization species in the gut symbiotic organ, even in a high inoculation competitiveness of the PBE and Pandoraea against the SBE was dose condition (Fig. 5B). Although the Pandoraea was compet- investigated. For this analysis, B. insecticola, B. fungorum, and itive against the SBE under a high (5,000 cells) infection dose, P. norimbergensis were used as representatives of SBE, PBE, and the SBE became significantly dominant under a lower (250 cells) Pandoraea, respectively. To investigate in vitro competitiveness, dose condition (Fig. 5B). Microscopic visualization of the com- log-phase cultured cells of the SBE (rifampicin resistance) and a petition process by use of an RFP-labeled B. insecticola (SBE) and competitor (PBE or Pandoraea [chloramphenicol resistance]) GFP-labeled B. fungorum (PBE) or P. norimbergensis (Pandoraea) were suspended in yeast-glucose (YG) medium, and incubated at revealed that, first the SBE and the nonnative bacteria cocolon- 25 °C without shaking for 2 d. The incubated medium was plated ized the M4 region in a mixed manner, that the SBE then grad- on YG agar plates containing either rifampicin or chloram- ually gained the upper hand, and that it eventually solely occupied phenicol, and the competitive index (CI) was calculated as (output the symbiotic organ in 5 d after coinoculation (Fig. 5 C and D). SBE CFU/input SBE CFU)/(output competitor CFU/input competitor CFU) (12). To determine the in vivo competitiveness, Similar results were consistently observed when the SBE species newly molted second-instar nymphs of R. pedestris were individu- was coinoculated with other PBE Burkholderia species (SI Ap- ally fed with 1 μL of distilled water containing the same CFUs of pendix, Fig. S11 and Table S3) and when another SBE species, the SBE and a competitor (B. fungorum or P. norimbergensis). Two Burkholderia cordobensis, was tested against the PBE and days after rearing, the symbiotic organs were dissected, their ho- Pandoraea species (SI Appendix, Fig. S12 and Table S3). These mogenates were plated on YG agar plates containing antibiotics, observations clearly demonstrate that the exclusive prevalence of and CI values were calculated based on the CFU counts. SBE Burkholderia in natural bean bug populations is supported While the SBE Burkholderia did not dominate the 2 other by the higher competitiveness of SBE species inside the gut species in vitro (Fig. 5A), the in vivo competitive assay revealed symbiotic organ.

Fig. 5. Infection competitiveness of symbiotic bacteria against nonnative bacteria. Competitiveness of SBE (B. insecticola) against PBE (B. fungorum), and Pandoraea (Pan: P. norimbergensis) in in vitro growth (A) and in vivo (B). Inoculum doses are indicated in CFU. Each dot represents a CI value from a microtube or an insect. The CI values are obtained by (output SBE colony count/input SBE colony count)/(output competitor colony count/input competitor colony count) and statistically evaluated by the 1-sample t test (against CI = 1.0). NS, not significant. (C and D) Visualization of the in vivo competition dynamics. An RFP- labeled SBE strain was coinoculated with a GFP-labeled strain of PBE (C)orPandoraea (D). dpi, days postinoculation. (Scale bars, 0.2 mm.)

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1912397116 Itoh et al. Downloaded by guest on September 30, 2021 Competitiveness in the Crypt Lumen Is Critical for the Riptortus– Burkholderia Specificity. Our previous studies have revealed that the bean bug R. pedestris develops a specific sorting organ called the “constricted region” in the midgut and employs in the M4 unique antimicrobial peptides to select bacteria (14, 15). This study confirmed that diverse bacterial species are winnowed out by the constricted region. However, the selective apparatus is not perfect, and bacteria that are closely related to the natural symbiont, including some PBE Burkholderia and Pandoraea species, can penetrate the constricted region and reach the gut symbiotic organ (Fig. 2 and SI Appendix, Table S2). Although the PBE and Pandoraea did not fully colonize the gut crypts (Fig. 3 and SI Appendix,Fig.S2), these nonnative bacteria stably accommodated in the M4 region and, surprisingly, were not harmful but, in contrast, beneficial for host survival, growth rate, and body size (Fig. 4). In contrast, SBE Burkholderia are highly adapted to the in- ternal environment of the gut symbiotic organ and always outcompete PBE and Pandoraea species (Fig. 5 and SI Appendix, Figs. S11 and S12). These results clearly demonstrated that the exclusive prevalence of SBE Burkholderia in natural bean bug populations is underpinned not only by host-mediated partner choice, but also by the higher competitiveness of SBE species inside the gut symbiotic organ. In symbiotic associations with environmental transmission, Fig. 6. Evolutionary hypothesis of the symbiotic traits in the Burkholderiaceae. host organisms have evolved elaborate mechanisms for partner Abilities for initiation, accommodation, and cooperation in the bean bug gut choice and sanctions of cheaters (4). Thus, to establish a specific have evolved in the common ancestor of the genera Pandoraea and association with their hosts, symbiotic microorganisms have to be Burkholderia (black box in the dendrogram), and only the competitiveness EVOLUTION capable of passing a sorting mechanism (initiation), colonizing in the gut symbiotic organ has evolved in the SBE lineage (blue box in the dendrogram). These traits, except the initiation ability, are lost in BCC&P the symbiotic organ (accommodation), and behaving coopera- (white box in the dendrogram). tively in the hosts (cooperation). Mutualists are generally char- acterized by these features. Strikingly, the inoculation of a broad range of Burkholderia and allied species revealed that these Possible Mechanisms Underpinning the Competitiveness. The mo- fundamental features of symbiotic microorganisms are not re- lecular mechanisms supporting the competitiveness of SBE in stricted to SBE but shared by PBE, and even the outgroup the gut symbiotic organ are at present unclear. Possible mechanisms Pandoraea, indicating that these symbiotic traits are ancestral for the competitiveness could be categorizedinto2groups:Direct (Fig. 6). In contrast, the advanced adaptation to and strong and indirect mechanisms. In the former mechanism, symbionts di- competitiveness in the gut symbiotic organ evolved exclusively rectly interfere with competitors, while in the latter mechanism, in the SBE lineage (Fig. 6). These features enable the SBE symbionts are selected by host-served specific conditions, such as Burkholderia to dominantly and stably occupy the symbiotic gut nutrients and antimicrobial agents. As for the former mechanism, – portion of the bean bug and underpin the host–symbiont speci- the T6SS is known as a competition factor in the squid Vibrio ficity. Previous studies on the SBE species B. insecticola have system and honey bee gut microbiota (42, 43). However, the T6SS revealed some of the genetic bases for the initiation, accom- of SBE Burkholderia is strongly repressed in vivo (17) and T6SS modation, and cooperation, all of which are conserved in PBE mutants outcompeted PBE and Pandoraea species, similarly to the and Pandoraea species: Flagellar motility for initiation (14), wild-type (SI Appendix,Fig.S13), strongly suggesting that the cT6SS polyhydroxyalkanoate/purine/cell wall/LPS biosynthesis for ac- is not involved in the in vivo competitiveness of SBE Burkholderia. In addition, SBE species lack the contact-dependent growth in- commodation (18, 35, 40, 41), and nitrogen and sulfur recycling for hibition system, which is also known as a microbe–microbe toxin cooperation (17). The extent to which differential activity of these delivery system in some Burkholderia (44). functions in competitive and noncompetitive strains contributes to Taking into account the histological observations, the latter, the colonization competitiveness will deserve attention in future indirect competition mechanism seems more plausible to the studies. bean bug system. The microscopic observations revealed that At this stage, it also remains unclear where and how the PBE and Pandoraea colonize and proliferate well in M4B and competitive traits have evolved in the SBE lineage. It is plausible the central tract of M4 but show a colonization defect inside that the competitive traits have been selected in the insect gut. crypts (Fig. 3), suggesting that the environment of the crypt lu- This scenario requires that the SBE Burkholderia gain fitness men is different from the central tract of the M4 and is highly through the symbiotic association, for example by an efficient specialized for housing SBE Burkholderia. This could contribute recolonization of the soil environment from the insect midgut to the in vivo competitiveness of the symbiont. Although spec- crypts. Although this aspect of the symbiosis is currently poorly ulative, SBE Burkholderia may efficiently utilize nutrients served investigated (10), recolonization of the soil does not seem im- by the host in the crypt lumen, and also adapt to specific chemical possible because in all stages of the insect life cycle, a high number conditions (e.g., pH, osmolarity, and oxygen concentration) of the of viable bacteria are present in the posterior midgut. Alterna- luminal environment. Since diverse crypt-specific antimicrobial tively, the competitive traits may have evolved due to selection on peptides are highly expressed (15), it is also plausible that SBE SBE Burkholderia lifestyles in a different environment, such as soil Burkholderia are more tolerant to the host gut immunity than the and rhizosphere, and the symbiont could outcompete other bac- less-competitive species. Furthermore, theoretical studies have teria in the M4 incidentally. To clarify this point, the symbiont’s pointed out that the adhesion ability is an additional pivotal char- benefit gained from the association should be investigated in acter to determine the competitiveness and stability of a bacterium forthcoming work. in complex microbiota developing on a surface substrate, such as

Itoh et al. PNAS Latest Articles | 7of10 Downloaded by guest on September 30, 2021 biofilms and intestinal flora (45). As an alternative mechanism, ent between each system (56, 57). The amazing diversity of sym- therefore, SBE Burkholderia may have an adhesin-mediated higher biotic lifestyles in this monophyletic group would provide a great affinity to the crypt epithelia. Future genome comparisons in opportunity to find out the different paths a microorganism can conjunction with gene-deletion or -transfer experiments could lead take to become a symbiont in the competitive microbial world. to the identification of key genes responsible for the SBE-specific colonization and competitiveness in the midgut crypts. Materials and Methods Insects and Bacterial Strains. The R. pedestris strain used in this study was Diversity of the Symbiont Specificity in Stinkbugs. In addition to originally collected from a soybean field in Tsukuba, Ibaraki, Japan, and the bean bug, other heteropteran species of the superfamilies maintained in the laboratory for over 10 y. The insects were reared in Petri Coreoidea and Lygaeoidea and the family Largidae are commonly dishes (90-mm diameter, 20-mm high) at 25 °C under a long-day regimen associated with Burkholderia in the midgut crypts. Although the (16-h light, 8-h dark) and fed with soybean seeds and distilled water con- bean bug and most members of the Coreoidea and Lygaeoidea are taining 0.05% ascorbic acid. Bacterial species/strains used in this study and their culture conditions are listed in SI Appendix, Tables S1 and S2. exclusively associated with SBE Burkholderia (32), some species are associated mainly with SBE but to a lesser extend also with – Oral Administration of Cultured Bacteria. PBE and even with BCC&P (46 50). In addition, members of the Taxonomically diverse bacteria. Bacterial species were grown in specific optimal Largidae harbor a specific group of PBE Burkholderia (51, 52). growth condition (SI Appendix, Table S1) to an early log phase on a gyratory

These previous surveys support the hypothesis that the symbiotic shaker at 150 rpm. Exponentially growing cells (approximately OD600 = 0.5) traits were ancestral in the genus Burkholderia,andalsosuggest were harvested by centrifugation and suspended in distilled water so that that the competition-based selection favors different Burkholderia the final concentration was 104 cells/μL. Newly molted second-instar nymphs clades in different stinkbug taxa. were deprived of water overnight to make the insects thirsty and willing to ingest the bacteria-containing suspensions. The nymphs were fed with Competition-Based Selection of an Optimal Symbiont. Theoretical symbiont-containing water for 48 h, after which it was replaced by sterile studies predicted the importance of host control (i.e., partner water. Two days after the third-instar molt (approximately 5 d after inoculation), the symbiotic organs were dissected under a binocular microscope choice and partner fidelity) for the evolution and maintenance of (S8APO, Leica), rinsed twice in sterilized water, and homogenized in steril- mutualistic associations (4, 53). Furthermore, more recent mod- ized water. Dilution series of the solution were then plated on agar plates to eling studies proposed competition-based mechanisms as an al- examine the infection status of the insects. If any colonies were detected, ternative to assure symbiont quality (8, 9, 45, 54), where the host the number of CFUs was calculated. serves as a selective environment to let a favorable microorganism Burkholderia and other Burkholderiaceae species. Burkholderia and other grow preferentially. Herein, we experimentally point out the im- Burkholderiaceae species were grown at 30 °C to an early log phase in YG portance of this mechanism in the Riptortus–Burkholderia symbi- medium(0.5%yeastextract,0.4%glucose,0.1%NaCl)forBurkholderia, otic system. Recent transcriptomic analyses revealed that SBE Pandoraea, Cupriavidus,andRalstonia, or R2A broth (Nippon Pharmaceu- Burkholderia recycle the host’s metabolic wastes into utilizable ticals) for Chitinimonas on a gyratory shaker at 150 rpm. Inoculations of the nutrients, such as essential amino acids and B vitamins (17). bacterial species were performed as described above. Two days after the third-instar molt, the symbiotic organs were dissected and examined for Combined with histological data, the gene-expression patterns in infection with the Burkholderia or other Burkholderiaceae species by di- the symbiotic organ further suggest that the Burkholderia symbiont agnostic PCR with specific primer sets for the Burkholderia 16S rRNA gene proliferates in the M4 crypts and that excess symbiont cells move (BF and BR) (58) or universal ones for the bacterial 16S rRNA gene (515F into the M4B region, where they are entirely digested and all of and 806R) (59), respectively, as previously described (32). the derived nutrients absorbed by the host (15, 17). In other words, B. mallei and B. pseudomallei. Inoculation tests of 2 BSL3 pathogens, B. mallei the bean bug cultivates the symbiont in the M4 and digests them and B. pseudomallei, were performed at the BSL3 facility of the Hokkaido in the M4B. In such a “cultivation symbiosis,” growth rate and University Research Center for Zoonosis Control. The 2 species were also regeneration efficiency of host metabolites in the symbiotic organ grown at 30 °C to an early log phase in YG medium on a gyratory shaker at seem to be directly linked to the host fitness, whereby the host 150 rpm, as well as other Burkholderia species, as described above. Oral administrations of the 2 species were performed as described above. Two could gain a significant benefit from the better grower. If so, we days after the third-instar molt, insects were preserved in acetone during could speculate that the host insect facilitates microbial competi- 1 wk so that eventual bacterial cells were completely killed. Total DNA was tion inside the symbiotic organ not only for winnowing out po- extracted from the abdominal part (without dissection) and subjected to tential parasites but also for selecting a better grower. In this diagnostic PCR to determine if the BCC&P species had colonized the gut context, it is notable that SBE showed greater benefits to the host symbiotic organ of R. pedestris. than PBE and Pandoraea species in terms of growth rate and body weight (Fig. 4). Moreover, the apparent absence of bacterial GFP- or RFP-Labeling of Bacterial Species. PBE species (B. fungorum, Burkholderia degradation in the M4B in PBE or Pandoraea infected insects (SI phytofirmans, Burkholderia graminis, Burkholderia kururiensis), BCC&P species Appendix, Fig. S3) could be a further reason for the reduced (B. plantarii, B. glumae, B. pyrrocinia), P. norimbergensis, E. coli,andC. benefits provided by these bacteria. taiwanensis were labeled with GFP by the Tn7 minitransposon system, as A competition-based symbiont selection also plays a pivotal previously described (14). For labeling SBE Burkholderia (B. insecticola and B. cordobensis) with red fluorescent protein (RFP), pMini-Tn7-kan-rfp (60) was role in the fungus-cultivating ambrosia beetles (55), suggesting used under the same experimental conditions. that in cultivation symbiosis, where hosts harvest growing symbionts, the competition-based mechanism is commonly impor- Histological Observations. Insects were inoculated with Burkholderia or tant not only for keeping symbiont quality but also maybe for Pandoraea species (listed in SI Appendix, Table S2) at the second instar, and selecting the better-growing symbionts. More generally, broader dissected 2 d after the third-instar molt (approximately 5 d after inocula- surveys could clarify the general impact of microbe–microbe tion). The whole midgut was dissected from each insect in PBS, and photo- competition in the evolution and stabilization of symbiotic as- graphed by a digital camera (EC3, Leica) connected to a dissection microscope sociations maintained via environmental symbiont transmission, (S8APO, Leica). To investigate colonization properties in more detail, GFP- as suggested by recent examples (42, 43). labeled B. insecticola (SBE), B. fungorum (PBE), and P. norimbergensis The genus Burkholderia (Burkholderia/Paraburkholderia/ (Pandoraea) were inoculated, and infected crypts were observed under a confocal LSM (TCS SP8, Leica). The dissected tissues were fixed with 4% Caballeronia) includes a variety of symbiotic lifestyles, such as the paraformaldehyde for 10 min at room temperature, washed in PBS twice, nodule symbionts in leguminous plants and farming symbionts in incubated in PBS containing 0.1% Triton X-100 for 5 min, stained with 0.5 μM the social amoeba, as well as the gut symbionts in stinkbugs, in of SYTOX Green, and 5 units/mL of Alexa Fluor555 phalloidin (Thermofisher) in which the molecular mechanisms underpinning the specificity (and PBS for 20 min, washed in PBS twice, and mounted on silane-coated glass slides probably importance of symbiont’s competitiveness) seem differ- for observations.

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1912397116 Itoh et al. Downloaded by guest on September 30, 2021 Competitive Infection Assays. To visualize the in vivo competition, an RFP-labeled SBE Burkholderia In vitro competition assay. For competition assays, a spontaneous rifampicin- (B. insecticola or B. cordobensis) and a GFP-labeled competitor (B. fungorum, mutant of B. insecticola (SBE) and spontaneous chloramphenicol-mutants B. phytofirmans, B. graminis, B. kururiensis,orP. norimbergensis)were of B. fungorum (PBE) and P. norimbergensis (Pandoraea) were used. Log-phase coinoculated, and the colonization process was investigated by fluorescence cultured cells of the SBE and a competitor (PBE or Pandoraea) were suspended microscopy (DMI4000B, Leica). Based on the microscope images, red and green in YG medium, adjusted to the same concentration at 5,000 CFU and mixed. fluorescent areas were calculated by ImageJ (61) and used to evaluate the The adjusted cells were diluted to 1 mL with YG medium in a 1.5-mL micro- competitiveness of the SBE Burkholderia species. tube, and incubated at 25 °C without shaking. Two days after the inoculation, To clarify whether T6SS is involved in the competition ability, 2 T6SS genes, the medium was serially diluted and plated on YG agar plates containing ei- vgrG and clpV (SI Appendix, Fig. S13A), were deleted in B. insecticola (SBE) ther rifampicin or chloramphenicol. After 3-d incubation at 25 °C, colonies of by the homologous recombination technique, as previously described (18). the SBE and competitor were counted on the plates. The CI was calculated vgrG, a tip protein playing a role as spike as well as an effector, is an essential + by (output SBE CFU/input SBE CFU)/(output competitor CFU/input competitor gene for functional T6SS (62). clpV,encodinganAAA ATPase, is also 1 of the CFU) (12). pivotal genes for the T6SS (62). The ΔvgrG and ΔclpV mutants were labeled In vivo competition assay. Log-phase cultured cells of the SBE and a competitor with RFP and subjected to the in vivo competition assay, as described above. (B. fungorum or P. norimbergensis) were suspended in distilled water, mixed, and adjusted to the same concentration at either 250 CFU/μL or 5,000 ACKNOWLEDGMENTS. We thank I. Nishi and N. Nakamura for technical CFU/μL infection dose. In each assay, newly molted second-instar nymphs of assistance; C. Matsuura and Y. Matsuura for beautiful artwork; H. Shimoji for R. pedestris were individually fed with 1 μL of the bacteria-containing sus- significant comments on statistics; and P. Mergaert for helpful comments pensions, as previously described (12). Insects were reared at 25 °C under a on this manuscript. This work has benefited from the BSL3 center of the long-day regimen and fed with soybean seeds and distilled water. Two days Hokkaido University Research Center for Zoonosis Control. This work was supported by Japan Society for the Promotion of Science Grants-in-Aid for after the inoculation, the symbiotic organ was dissected from each of the Scientific Research 19K15724 (to H.I.) and Ministry of Education, Culture, insects and homogenized with sterile distilled water. The output homoge- Sports, Science, and Technology Grants-in-Aid for Scientific Research nate was serially diluted and plated on YG agar plates containing antibiotics. 15H05638 (to Y.K.), the Canon foundation (H.I.), the Japan Society for the After 3-d incubation at 25 °C, colonies of the SBE and competitor were Promotion of Science (JSPS) Bilateral Joined Research Project (Y.K.), and JSPS counted on the plates and CI values were calculated. Research Fellowship for Young Scientists 201911493 (to S.J.).

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